The epigenetic modification of cytosine in DNA is essential for normal vertebrate development yet adds a substantial mutagenic burden on the organism. This project seeks to understand the mechanisms of mutation at CpG sites and to determine how methylation patterns change during transformation and contribute to gene inactivation. Sequencing of mutations in tumor suppressor genes such as p53 in human cancers has shown that CpG methylation sites are common sites for mutations in somatic cells, as well as in the human germline. DNA methylation therefore contributes very significantly to the generation of human point mutations and these mutations are responsible, in some cases, for the activation of tumor suppressor genes. We will investigate methylation changes in human bladder cancer since our preliminary studies have already identified some of the key genetic changes involved in the multistep transformation of human uroeptithelium. New genetic changes involved in the multistep transformation of human uroepithelium. New genetic assays we have established, will be used to fully define the role of DNA methylation in generating CT transition mutations. In particular, we will follow-up our observation that the DNA methyltransferase itself is capable of inducing a high percentage of transitions. Since we are interested in understanding the role of methylation in controlling the expression of vertebrate genes, we will also use a model system we have derived to investigate the molecular mechanisms of genomic imprinting. A somatic cell line has been established which contains a maternal duplication/paternal deficiency of Igf2 which can be induced to express Igf2 by 5-azacytidine or BudR treatment. Our goals are therefore to determine how methylation patterns are established, their roles in suppressing the activities of certain genes during differentiation and transformation and to determine the significance of this process in the generation of human mutations.